The smooth muscle of rat aorta was used as a model for the study of the molecular mechanisms of 5-HT2 receptor function. We have previously shown that 5-HT receptors in rat aorta are coupled to phosphoinositide (PI) - specific phospholipase C. Further, we showed that the mechanism of contraction elicited by 5-HT is a complicated scenario involving receptor-mediated activation of calcium channels and a phospholipase C. We now report that in rat aorta, the 5-HT-induced contraction and PI turnover are modulated by biologically active phorbol esters. In rat aorta, 5-HT stimulated PI turnover and contraction (EC50=10 plus or minus 3 uM); these two responses were highly correlated (r=0.95; P less than .01). We have characterized the inhibitory potency of a variety of 5-HT-antagonists against the stimulation of P1 turnover elicited by 5-HT. Classic 5-HT2 anatagonists mianserin, ketanserin, metergoline and pizotifen were found to inhibit this response in the low nanomolar range; amitryptiline and haloperidol were 10- to 20- fold less potent. The alpha-1 receptor antagonist, prazosin, was inactive in micromolar concentrations. The potency of the 5-HT2 antagonists was correlated with their ability to displace (3H) ketanserin binding from rat frontal cortex membranes (r=0.90; P less than .05). The tumor promoter phorbol dibutyrate was found to inhibit 5-HT-stimulated PI turnover at low nanomolar concentrations whereas the biologically inactive substance 4-alpha-phorbol was ineffective. Pretreatment of rat aorta with phorbol dibutyrate at concentrations that inhibited 5- HT-induced PI turnover also attenuated the aortic contraction induced by 5-HT in the presence of a calcium channel blocker nitrendipine. Our results suggests that phorbol esters may densensitize 5-HT2-receptor-mediated PI turnover and contraction of rat aorta, possibly via an activation of protein kinase C.